Papel do ferro no mecanismo fungicida mediado pelo óxido nítrico de macrófagos murinos ativados com IFN-gama contra conídias do Paracoccidioides brasiliensis

Iron is an essential growth element of virtually all microorganisms and its restriction is one of the mechanisms used by macrophages to control microbial multiplication. Paracoccidioides brasiliensis, the agent of paracoccidioidomycosis, an important systemic mycosis in Latin America, is inhibited in its conidia-to-yeast conversion in the absence of iron. We studied the participation of iron in the nitric oxide (NO)-mediated fungicidal mechanism against conidia. Peritoneal murine macrophages activated with 50U/mL of IFN-gamma or treated with 35 µM Deferoxamine (DEX) and infected with P. brasiliensis conidia, were co-cultured and incubated for 96 h in the presence of different concentrations of holotransferrin (HOLO) and FeS0(4). The supernatants were withdrawn in order to assess NO2 production by the Griess method. The monolayers were fixed, stained and observed microscopically. The percentage of the conidia-to-yeast transition was estimated by counting 200 intracellular propagules. IFN-gamma-activated or DEX-treated Mthetas presented marked inhibition of the conidia-to-yeast conversion (19 and 56%, respectively) in comparison with non-activated or untreated Mthetas (80%). IFN-gamma-activated macrophages produced high NO levels in comparison with the controls. Additionally, when the activated or treated-macrophages were supplemented with iron donors (HOLO or FeSO4), the inhibitory action was reversed, although NO production remained intact. These results suggest that the NO-mediated fungicidal mechanism exerted by IFN-gamma-activated macrophages against P. brasiliensis conidia, is dependent of an iron interaction.O ferro é elemento essencial para o crescimento de microrganismos e sua limitação é um dos mecanismos usados por macrófagos para controlar a multiplicação microbiana. Paracoccidioides brasiliensis, o agente da paracoccidioidomicose, uma das micoses sistêmicas mais importantes na América Latina, é inibido em sua conversão de conídia-à-levedura na ausência do ferro. Estudamos a participação do ferro no mecanismo fungicida mediado pelo óxido nítrico (NO) na sua interação com as conídias do fungo. Macrófagos peritoneais murinos ativados com 50U/mL de IFN-gama ou tratados com 35 µM Deferoxamina (DEX) e infectados com conídias do P. brasiliensis foram co-cultivados e incubados por 96 h na presença de concentrações diferentes de holotransferrina (HOLO) e FeS0(4). Os sobrenadantes foram retirados a fim de avaliar a produção de NO2 pelo método de Griess. Os macrófagos eram fixados, corados e observados ao microscópio. A porcentagem da transição de conídia-à-levedura foi estimada contando 200 propágulos intracelulares. Os macrófagos ativados com citocina ou tratados com DEX apresentaram inibição marcada da conversão de conídia-à-levedura (19 e 56%, respectivamente) em comparação com macrófagos controle (80%). Os macrófagos ativados com IFN-gama produziram elevação nos níveis de NO em comparação com macrófagos não-tratados ou não-activados. Adicionalmente, quando as monocapas ativadas ou tratadas foram suplementadas com doadores do ferro (HOLO ou FeSO4), a ação inibitória foi revertida embora a produção de NO permanecesse intacto. Estes resultados sugerem que o mecanismo fungicida mediado pelo NO exercido por macrófagos ativados com IFN-gama contra conídias do P. brasiliensis é dependente de uma interação do ferro

Purificacion de antigenos somaticos del Paracoccidioides brasiliensis. Estudio preliminar

We describe the procedures followed to purify antigenic fractions from P. brasiliensis sonicated whole yeast cells. By means of the agar gel immunodiffusion test, antigen-antibody complexes were allowed to produce precipitin lines; these were further separated, eluated and two antibody-free antigenic fractions were further purified by column chromatography and polyacrilamyde gel electrophoreses. These fractions proved to be proteins with molecular weights of 66 and 85 kd; the former reacted with specific antisera and produced a precipitin line identical to one of the three formed by the total antigen. Results indicate that it would feasible to obtain purified, chemically characterized antigens. These could, in the future, bring improvement in the serologic diagnosis of paracoccidioidomycosis as only those specific components of known serologic reactivity would be employed.Se describen los procedimientos de purificación empleados para la separación de las fracciones antigénicas a partir de un material somático obtenido por rotura de células levaduras completas de P. brasiliensis. Dichas fracciones mostraron ser proteínas con pesos moleculares de 66 y 85 Kd; la primera de ellas reaccionó con sueros específicos produciendo una banda de precipitado idéntica a una de las 3 desarrolladas por el antígeno total. Los resultados señalan la posibilidad de obtener antígenos purificados, químicamente identificados y cuyo uso pudiera, en el futuro, representar ventajas para el diagnóstico serológico de la paracoccidioidomicosis, permitiendo separar, repetidamente, solo aquel componente reconocidamente activo

Sequela fibrótica na paracoccidioidomicose pulmonar: aspectos histopatológicos em camundongos BALB/c infectados com propágulos viáveis e não viáveis do Paracoccidioides brasiliensis

Patients with paracoccidioidomycosis often present pulmonary fibrosis and exhibit important respiratory limitations. Based on an already established animal model, the contribution of viable and non-viable P. brasiliensis propagules to the development of fibrosis was investigated. BALB/c male mice, 4-6 weeks old were inoculated intranasally either with 4x10(6 )viable conidia (Group I), or 6.5x10(6) fragmented yeast cells (Group II). Control animals received PBS. Six mice per period were sacrificed at 24, 48, 72h (initial) and 1, 2, 4, 8, 12 and 16 weeks post-challenge (late). Paraffin embedded lungs were sectioned and stained with H&E, trichromic (Masson), reticulin and Grocott´s. During the initial period PMNs influx was important in both groups and acute inflammation involving 34% to 45% of the lungs was noticed. Later on, mononuclear cells predominated. In group I, the inflammation progressed and granulomas were formed and by the 12th week they fussed and became loose. Thick collagen I fibers were observed in 66.6% and 83.3% of the animals at 8 and 12 weeks, respectively. Collagen III, thick fibers became apparent in some animals at 4weeks and by 12 weeks, 83% of them exhibited alterations in the organization and thickness of these elements. In group II mice, this pattern was different with stepwise decrease in the number of inflammatory foci and lack of granulomas. Although initially most animals in this group had minor alterations in thin collagen I fibers, they disappeared by the 4th week. Results indicate that tissue response to fragmented yeast cells was transitory while viable conidia evoked a progressive inflammatory reaction leading to granuloma formation and to excess production and/or disarrangement of collagens I and III; the latter led to fibrosis.Pacientes com paracoccidioidomicose apresentam, algumas vezes, fibrose pulmonar e exibem limitações respiratórias importantes. Baseados num modelo animal já estabelecido da micose, estudamos a possível contribuição de propágulos viáveis e não viáveis do Paracoccidioides brasiliensis ao desenvolvimento da fibrose. Assim, camundongos BALB/c, machos de 4 a 6 semanas de idade, foram inoculados intranasalmente com 4 x 10(6) conídios viáveis (Grupo I), ou com 6,5 x 10(6) fragmentos não viáveis de células leveduriformes (Grupo II). Animais controles (Grupo III) receberam unicamente PBS. Seis camundongos por período foram sacrificados 24, 48, 72h (inicial) e 1, 2, 4, 8, 12 e 16 semanas pós-inoculação (tardio). Os pulmões dos animais foram fixados, incluidos em parafina, cortados e corados com H & E, Tricrômico (Masson), reticulina e Grocott. Durante o período inicial houve afluxo importante de PMNs em ambos os grupos I e II, e a inflamação aguda comprometeu entre 34 a 45% dos pulmões. Depois, foram as células mononucleares as que predominaram. No grupo I, a inflamação progrediu e formaram-se granulomas os quais, às 12 semanas, ficaram confluentes e frouxos. Adicionalmente, se observaram fibras de colágeno tipo I muito densas em 66,6% e 83,3% dos animais após 8 e 12 semanas, respectivamente. As fibras do colágeno tipo III foram observadas nos animais a partir das 4 semanas pós-infecção, e 83% deles exibiram, às 12 semanas, alterações na sua distribuição e organização. Nos animais do grupo II o padrão foi diferente, pois mostraram diminuição gradual no número de focos inflamatórios e não houve formação dos granulomas. Embora animais deste grupo tivessem no período inicial pequenas alterações nas fibras de colágeno tipo I, estas desapareceram por volta da 4a semana. Os resultados indicam que a resposta tissular aos fragmentos de leveduras foi transitória, enquanto que os conídios induzem resposta inflamatória progressiva permitindo a formação de granuloma e um excesso na produção e desorganização dos colágenos I e III, permitindo finalmente a fibrose

Análisis de concordancia de diferentes metodologías para la identificación de aislamientos orales de especies de candida

Background: The yeasts species determination is fundamental not only for an accurate diagnosis but also for establishing a suitable patient treatment. We performed a concordance study of five methodologies for the species identification of oral isolates of Candida in Colombia. Methods: Sixty-seven Candida isolates were tested by; API® 20C-AUX, Vitek®2 Compact, Vitek®MS, Microflex® and a molecular test (panfungal PCR and sequencing). The commercial cost and processing time of the samples was done by graphical analysis. Results: Panfungal PCR differentiated 12 species of Candida, Vitek®MS and Microflex® methods identified 9 species, and API® 20C-AUX and Vitek®2 Compact methods identified 8 species each. Weighted Kappa (wK) showed a high agreement between Panfungal PCR, Vitek®MS, Microflex® and API® 20C-AUX (wK 0.62-0.93). The wK that involved the Vitek®2 Compact method presented moderate or good concordances compared with the other methods (wK 0.56-0.73). Methodologies based on MALDI TOF MS required 4 minutes to generate results and the Microflex® method had the lowest selling price. Conclusion: The methods evaluated showed high concordance in their results, being higher for the molecular methods and the methodologies based on MALDI TOF. The latter are faster and cheaper, presenting as promising alternatives for the routine identification of yeast species of the genus Candida. © 2018. Universidad del Valle

Estandarización y validación de un método de cromatografía líquida de alta eficiencia con detector de arreglo de diodos (HPLC-DAD) para la determinación de niveles sanguíneos de voriconazol

Introduction. A specialized service for the determination of antifungal blood levels is not available in Colombia, this service is essential for the proper follow-up of the antifungal therapy.Objective. The aim of this study was to standardize and validate a simple, sensitive, and specific protocol, based on High Performance Liquid Chromatography with Diode Array Detector (HPLC-DAD) for the quantification of voriconazole blood levels. Materials and methods. An Agilent HPLC-series-1200 equipment with UV-DAD detector was used. Analytical column-Eclipse XDB-C18 and pre- column Eclipse-XDB-C18, Agilent were also used. We used voriconazole as the primary control, and posaconazole as an internal control. The validation was done following the Food and Drug Administration (FDA) recommendations.Results. Best chromatographic conditions were: Column temperature of 25°C, UV variable wavelength detectors (VWD) detection at 256 nm for voriconazole, and 261 nm for Posaconazole (internal standard), 50 μL of injection volume, flow of volume 0,8mL/min, time of run of 10 min, mobile phase of acetonitrile:water (60:40). Finally, retention times were 3.13 and 5.16 min for the voriconazole and posaconazole, respectively. Range of quantification ranging from 0.125 μg/mL to 16 μg/mL. Conclusion. The selectivity and chromatographic purity of the obtained signal as well as the limits of detection and quantification standardized make this method an excellent tool for therapeutic monitoring of patients treated with voriconazole.Introducción. Hasta la fecha en Colombia no contábamos con un servicio especializado de medición de niveles séricos de antifúngicos, procedimiento esencial para el adecuado manejo del tratamiento de las Infecciones Fúngicas Invasoras (IFI). Objetivo. Estandarizar y validar un protocolo, simple, sensible y específico, basado en Cromatografía Líquida de Alta Eficiencia con detector de arreglo de diodos (HPLC-DAD) para la cuantificación de los niveles séricos de voriconazol. Materiales y métodos. Se usó un equipo HPLC-Agilent, serie-1200, con detector UV-DAD columna analítica Eclipse-XDB-C18 y una pre-columna Eclipse-XDB-C18, ambas de la marca Agilent. Como control primario se utilizó voriconazol, y como control interno posaconazol. La validación se hizo cumpliendo todos los criterios de aceptación de los parámetros recomendados por la Food and Drug Administration (FDA). Resultados. Las mejores condiciones cromatográficas se obtuvieron bajo los siguientes parámetros: temperatura de la columna de 25°C, detección UV-VWD de 261 nm, volumen de inyección de 50 μL, flujo de 0,8mL/min y un tiempo de corrido de 10 min. La fase móvil usada fue acetonitrilo:agua (40:60), obteniendo finalmente unos tiempos de retención de 3,13 y 5,16 min para el voriconazol y posaconazol respectivamente. El rango de cuantificación fue desde 0,125 μg/mL hasta 16 μg/mL. Conclusiones. La selectividad y pureza de la señal cromatográfica obtenida, así como los límites de detección y cuantificación estandarizados, hacen de esta metodología una excelente herramienta para el seguimiento terapéutico de los pacientes bajo tratamiento o profilaxis con voriconazol

Concordance analysis between different methodologies used for identification of oral isolates of Candida species

Introducción: La clasificación a nivel de especies de las levaduras del género Candida de origen clínico es fundamental para el diagnóstico y la instauración de un adecuado tratamiento para el paciente. Se realizó un estudio de concordancia de cinco metodologías usadas para la identificación de aislamientos orales de Candida spp en Colombia. Métodos: Sesenta y siete aislamientos de Candida spp fueron identificados a nivel de especie utilizando; API® 20 C AUX‚ Vitek® 2 Compact, MALDI TOF (Vitek® MS y Microflex®) y una prueba molecular, PCR Panfungal y secuenciación. Un análisis del costo comercial y tiempo de procesamiento de las muestras por cada método fue realizado mediante el análisis gráfico de ambas variables. Resultados: La PCR Panfungal y secuenciación diferenció 12 especies de Candida ‚ los métodos Vitek® MS y Microflex® identificaron 9 especies y los métodos API® 20 C AUX y Vitek® 2 Compact identificaron 8 especies. El análisis de Kappa ponderado (wK) demostró una concordancia alta entre los métodos PCR Panfungal y secuenciación‚ Vitek® MS‚ Microflex® y API® 20 C AUX‚ concordancias agrupadas en las categorías buena y muy buena (wK 0.62-0.93); los Kp que involucraron el método Vitek® 2 Compact presentaron concordancias moderadas o buenas frente a los otros métodos (wK 0.56-0.73). Las metodologías basadas en MALDI TOF MS requirieron 4 minutos para generar un resultado y el método Microflex® fue el método que en nuestro medio presentó el menor precio de venta del servicio. Conclusión: Los métodos evaluados presentaron una alta concordancia en sus resultados‚ siendo más alta para los métodos moleculares y las metodologías basadas en MALDI TOF MS; estas últimas son metodologías más rápidas, económicas y precisas, las cuales se presentan como alternativas prometedoras para la identificación rutinaria de especies de levaduras del género Candida.Q3Q3Artículo original160-167Background: The yeasts species determination is fundamental not only for an accurate diagnosis but also for establishing a suitable patient treatment. We performed a concordance study of five methodologies for the species identification of oral isolates of Candida in Colombia. Methods: Sixty-seven Candida isolates were tested by; API® 20C-AUX, Vitek®2 Compact, Vitek®MS, Microflex® and a molecular test (panfungal PCR and sequencing). The commercial cost and processing time of the samples was done by graphical analysis. Results: Panfungal PCR differentiated 12 species of Candida, Vitek®MS and Microflex® methods identified 9 species, and API® 20C-AUX and Vitek®2 Compact methods identified 8 species each. Weighted Kappa (wK) showed a high agreement between Panfungal PCR, Vitek®MS, Microflex® and API® 20C-AUX (wK 0.62-0.93). The wK that involved the Vitek®2 Compact method presented moderate or good concordances compared with the other methods (wK 0.56-0.73). Methodologies based on MALDI TOF MS required 4 minutes to generate results and the Microflex® method had the lowest selling price. Conclusion: The methods evaluated showed high concordance in their results, being higher for the molecular methods and the methodologies based on MALDI TOF. The latter are faster and cheaper, presenting as promising alternatives for the routine identification of yeast species of the genus Candida

Factores de riesgo para trastornos de la alimentación en los alumnos de la Universidad de Manizales

ResumenObjetivo: Determinar la frecuencia de los factores de riesgo para trastornos de la alimentación en estudiantes de la Universidad de Manizales. Materiales y métodos: De 3,610 estudiantes regulares de la Universidad  de Manizales se tomó una muestra de 165 estudiantes, quienes respondieron un cuestionario integrado compuesto por variables demográficas, las escalas de Zung para ansiedad o depresión,el Eating Disorderrs Inventory (EDI2) y el Apgar familiar; además se tomaron las medidas de peso, talla, índice de masa corporal y pliegue en el antebrazo. Resultado: Un12.7% de la población presentó factor de riesgo positivo para trastornos de la conducta alimentaría, 17.3% de la población femenina y el 3.8% de la población masculina; las personas con mayor factor de riesgo fueron las mujeres en la facultad de Comunicación Social y Periodismo (24.1%). Se determinó una  relación significativa entre la variable impulso por la delgadez con la ansiedad o depresión. El mayor índice de masa corporal y de porcentaje de grasa está asociado con un aumentoen la variable impulso por la delgadez. Conclusiones:Se encontraron factores de riesgo asociados con trastornos de la alimentación. La frecuencia del factor de riesgo según EDI2 en este estudio fue más baja que la encontrada en estudios realizados en Medellín y Bogotá.[Cano AA, Castaño JJ, Corredor DA, García AM, González M, Lloreda OL, et al. Factores de riesgo para trastornos de la alimentación en los alumnos de la Universidad de Manizales. MedUNAB 2007; 10:187-194].Palabras claves: Trastornos de la conducta alimentaria, factores de riesgo; Anorexia nerviosa, Bulimia